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Objective <p>To investigate the functional heterogeneity of adipose-derived stem cells (ASCs) in fat grafting and develop a density-based separation method to isolate distinct ASC subpopulations for improving graft survival.</p> Methods <p>ASCs were isolated from human lipoaspirates and subjected to adipogenic differentiation. A novel Percoll density gradient centrifugation protocol (10–100%, 300 × <i>g</i>, 35 min) was developed to separate ASCs into upper-layer (U-ASCs) and lower-layer (D-ASCs) subpopulations. Their adipogenic and angiogenic potentials were assessed through Oil Red O staining and Matrigel tube formation assays. In vivo functionality was evaluated in nude mice (<i>n </i>= 84) receiving fat grafts supplemented with GFP-labeled U-ASCs, D-ASCs, or unsorted ASCs (<i>n </i>= 28/group), with graft retention, histology, and cellular fate analyzed over 12 weeks.</p> Results <p>Density separation yielded two functionally distinct populations: U-ASCs exhibited 3.2-fold greater lipid accumulation (<i>p </i>&lt; 0.001), while D-ASCs showed 2.7-fold enhanced tube formation (<i>p </i>&lt; 0.01). In vivo, D-ASCs grafts demonstrated 58.3 ± 6.7% retention at 12 weeks versus 32.1 ± 5.2% for U-ASCs (<i>p </i>&lt; 0.001), with significantly reduced oil cysts (<i>p </i>&lt; 0.05) and 2.4-fold higher vascular density (<i>p </i>&lt; 0.001). Immunofluorescence revealed U-ASCs primarily differentiated into adipocytes (GFP+/Perilipin+), whereas D-ASCs integrated into vasculature (GFP+/CD31+).</p> Conclusion <p>ASC subpopulations differentially regulate fat graft outcomes—U-ASCs drive adipogenesis while D-ASCs enhance vascularization. The developed density separation method enables isolation of therapeutic ASC subsets, with D-ASCs showing superior potential for improving graft survival through neovascularization. These findings support precision strategies in cell-assisted lipotransfer.</p> Level of Evidence I <p>This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors <a href="http://www.springer.com/00266">www.springer.com/00266</a>.</p>

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Functional Stratification of Adipose-Derived Stem Cells via Gradient Density Centrifugation: D-ASCs Drive Vascularization for Enhanced Fat Graft Survival

  • 期刊论文

  • 发表日期：

    2026年5月4日

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  • Jie Long,
  • Guo Chen,
  • Zijin Qin,
  • Changxin Jin,
  • Hengxin Liu,
  • Jie Chen,
  • Baoqiang Song,
  • Ziang Zhang

摘要

Objective

To investigate the functional heterogeneity of adipose-derived stem cells (ASCs) in fat grafting and develop a density-based separation method to isolate distinct ASC subpopulations for improving graft survival.

Methods

ASCs were isolated from human lipoaspirates and subjected to adipogenic differentiation. A novel Percoll density gradient centrifugation protocol (10–100%, 300 × g, 35 min) was developed to separate ASCs into upper-layer (U-ASCs) and lower-layer (D-ASCs) subpopulations. Their adipogenic and angiogenic potentials were assessed through Oil Red O staining and Matrigel tube formation assays. In vivo functionality was evaluated in nude mice (n = 84) receiving fat grafts supplemented with GFP-labeled U-ASCs, D-ASCs, or unsorted ASCs (n = 28/group), with graft retention, histology, and cellular fate analyzed over 12 weeks.

Results

Density separation yielded two functionally distinct populations: U-ASCs exhibited 3.2-fold greater lipid accumulation (p < 0.001), while D-ASCs showed 2.7-fold enhanced tube formation (p < 0.01). In vivo, D-ASCs grafts demonstrated 58.3 ± 6.7% retention at 12 weeks versus 32.1 ± 5.2% for U-ASCs (p < 0.001), with significantly reduced oil cysts (p < 0.05) and 2.4-fold higher vascular density (p < 0.001). Immunofluorescence revealed U-ASCs primarily differentiated into adipocytes (GFP+/Perilipin+), whereas D-ASCs integrated into vasculature (GFP+/CD31+).

Conclusion

ASC subpopulations differentially regulate fat graft outcomes—U-ASCs drive adipogenesis while D-ASCs enhance vascularization. The developed density separation method enables isolation of therapeutic ASC subsets, with D-ASCs showing superior potential for improving graft survival through neovascularization. These findings support precision strategies in cell-assisted lipotransfer.

Level of Evidence I

This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.